Analysis of Renal Responses to Whole-body γ-Irradiation alone or Combined with Skin Injury Before the Onset of Renal Dysfunction in Mice

1. Introduction

Ionizing radiation injuries (RI), often in conjunction with other injuries namely combined injury (CI), have historically and contemporarily manifested in both warfare scenarios, such as Hiroshima and Nagasaki, and nuclear accidents like Chernobyl and Fukushima. The persistent relevance of these events underscores the need for a deep understanding of the biological impacts of such injuries. RI and CI manifest across a spectrum of symptoms classified temporally into acute, consequential, and late effects. Acute effects occur during or immediately after exposure, consequential effects evolve from ongoing damage after the initial exposure, and late effects, such as kidney injury, may not surface until months or years later [1,2,3,4,5,6,7].

The pathophysiology of radiation-induced kidney damage is complex and multi-faceted. Key mechanisms include direct DNA damage, inflammation, oxidative stress, microvascular damage, mitochondrial impairment, all of which culminate in renal tubular cell death [3,8,9,10,11,12]. There is no specific treatment for radiation-induced kidney injury or any types of acute kidney injury (AKI) [13,14].

Transcription factors like STAT1 and STAT3 are instrumental in regulating inflammatory mediators. They are activated by phosphorylation of certain amino acids, including tyrosine residuals. Protein tyrosine phosphatase SHP-1 modulates these pathways by dephosphorylating STAT proteins, thus acting as a critical regulatory checkpoint [15,16,17]. Ionizing radiation induces reactive oxygen species (ROS), which include superoxide anion and hydrogen peroxide. The body's defense against ROS involves several antioxidative enzymes, including heme oxygenase-1 (HO-1), superoxide dismutase (SOD), catalase, and peroxidase [18,19,20,21,22]. HO-1, in particular, is crucial for mitigating oxidative stress by breaking down heme, producing antioxidants like biliverdin and bilirubin, generating the protective gas carbon monoxide, and preventing iron from contributing to oxidative damage [23,24,25]. The interplay between inflammation and ROS can establish a self-reinforcing cycle, where immune cells augment ROS levels, which then escalates further inflammation, thereby exacerbating organ injury [20,26]. AKT, also known as protein kinase B (PKB), is a serine/threonine kinase and plays a protective role in organ injury by promoting cell survival, reducing apoptosis, enhancing cell proliferation, suppressing inflammation, and preserving mitochondrial function [27,28]. AKT often inhibits p53 in order to exert its pro- cell survival and anti-apoptotic cell death function [29,30,31].

Although the pathophysiology of RI and CI-induced kidney damage has become increasingly understood, the molecular pathways affected before any noticeable functional kidney damage remain largely unknown. Investigating these early alterations offers insight into the radiation's effects at the cellular and molecular levels, potentially elucidating why the kidney is resilient to immediate radiation-induced injury and identifying new therapeutic targets for intervention. In this study, we explored and contrasted the molecular pathways associated with inflammation, oxidative stress, and cell survival 30 days after mice were injured by radiation alone or in combination with a skin wound.

2. Results

Radiation alone or in combination with a skin wound has little immediate effect on renal function. As expected, RI did not significantly increase BUN levels (Figure 1a), serum NGAL levels (Figure 1b), or protein abundance of KIM-1 (Figure 1c), a marker for renal tubular damage, in the mouse renal cortex compared to the sham group 30 days after mice were exposed to 9.5 Gy of ⁶⁰Co radiation. CI did not significantly affect BUN or NGAL levels (Figure 1d and e), but increased KIM-1 protein abundance (Figure 1f) in the renal cortex when compared to the wound group. Compared with the sham group, wound alone had no significant effects on BUN (sham 42.7±2.8 mg/dL vs. wound 35.2±3.0 mg/dL) or serum NGAL levels (sham 3.2±0.7 ng/mL vs. wound 2.1±0.7 ng/mL) (Figure 1a, b, d and e). These data suggest that neither RI nor CI reduce kidney function within 30 days after injury, but CI has a nuanced effect at the molecular level.

Radiation alone and combined injury upregulate inflammatory pathways, but they do so in distinct ways in the renal cortex. While RI had no significant effect on STAT1-Y701 phosphorylation (Figure 2a), it significantly increased STAT3-Y705 phosphorylation compared with the sham group (Figure 2b). In contrast, CI significantly increased STAT1-Y701 phosphorylation (Figure 2c), but it had no significant effects on STAT3-Y705 phosphorylation (Figure 2d). Wound alone significantly increased phosphorylation of both STAT1-Y701 and STAT3-Y705 compared with the sham group (Figure 2e and f). Having found that RI, CI and wound alone activated STAT3 and STAT1, we sought to determine whether the insults affected the upstream signaling pathway of STATs. None of the insults had a significant effect on SHP-1-S591 phosphorylation or protein abundance of SHP-1 (Figure 2g to i). However, RI decreased p38 phosphorylation (Figure 2j), whereas CI tended to increase p38 protein abundance (Figure 2k). Wound alone had no significant effects on phosphorylation of p38 or p38 protein abundance when compared with the sham group (Figure 2l). These data indicate that RI and CI activate inflammatory pathways through different mechanisms without SHP-1 involvement in either pathway.

Radiation alone and combined injury have different effects on pro- and anti-inflammatory gene mRNA levels in the renal cortex. We found that while RI and CI had no significant effects on the majority of pro-inflammatory gene mRNA levels that were examined, RI significantly up-regulated mRNA abundance of IFNγR1, IFNγR2, and heparanase compared with the sham group (Figure 3a), whereas CI only tended to increase mRNA levels of heparanase (Figure 3b). Likewise, RI and CI had different effects on mRNA levels of immune-regulatory genes. RI remarkably increased IL-10 mRNA levels by 287.2%, whereas CI had no significant effects on the IL-10 mRNA abundance, but reduced the IL-4 mRNA abundance by 61.5% compared with their respective controls (Figure 3c and d). Wound alone did not notably impact the mRNA levels of most pro-inflammatory and immune-regulatory genes. However, it led to a significant decrease in IFNγR1 mRNA levels when compared to the sham group, as shown in Figure 3e and f.

Radiation alone and combined injury upregulate HO-1. Both RI and CI increased the HO-1 protein abundance by 80.7% and 38.3%, respectively, in the renal cortex when compared to their respective control groups, indicating the presence of oxidative stress after injuries (Figure 4a and b). However, RI had an additional effect of reducing the protein abundance of MnSOD and catalase (Figure 4c and g), whereas CI did not exhibit this reduction (Figure 4d and h). To gain a comprehensive understanding of how these two injuries affected the anti-oxidative stress pathways, we conducted additional experiments to assess their impact on CuZnSOD, PRDX1, and ferritin heavy chain. Our findings indicate that neither RI nor CI had a significant effect on the levels of these proteins (Figure 4e, f, and i to l). When compared with the sham group, wound alone had no significant effects on these anti-oxidant protein levels except for reducing catalase protein abundance (Figure 4p).

Radiation alone and combined injury upregulate cell survival and downregulate cell apoptosis pathways. Both RI and CI led to a remarkable increase in the phosphorylation of AKT-S473 (149.0% and 208.8% respectively, Figure 5a and b), while CI reduced protein abundance of AKT1 and AKT2 compared with wound alone (Figure 5b). However, RI and CI had different effects on the phosphorylation of GSK-3β-S9, one of AKT's downstream targets. While RI increased the phosphorylation of GSK-3β-S9 and concurrently decreased the overall GSK-3β protein levels, CI did not significantly affect either parameter, highlighting the complexity of AKT signaling pathways (Figure 5c and d). Despite any changes in protein abundance of HSC-70 (heat shock cognate protein 70), a pro-survival molecule (Figure 5e and f), both injuries resulted in a decrease in protein abundance of p53 when compared with their respective controls (Figure 5g and h). Wounding by itself did not significantly alter the levels of any of these proteins in comparison to the sham group (Figure 5i to l).

3. Discussion

4. Methods and Materials

Animal and experimental design. All procedures were approved by the Institutional Animal Care and Use Committee of Uniformed Services University (MED-22-099). B6D2F1/J female mice (12 weeks old, approximately 20-26 g) purchased from Jackson Laboratory (Bar Harbor, ME) were maintained in a facility accredited by AAALAC in plastic microisolator cages with hardwood chip bedding and allowed to acclimate to their surroundings for at least 3 days prior to the study. Male mice were not used in this study because of potential problems associated with male mouse aggression, such as fight wounds which were not desirable during the experimental period. Previous injury studies [57] also used female mice for this reason. As such, we continued to conduct this study with female mice so that data collected could be compared with previous ones. These mice were provided with commercial rodent chow (Rodent Diet #8604, Harlan Teklad, Madison, WI) and acidified tap water (pH=2.5-2.8) ad libitum. Rooms holding animals were maintained at 22°C ± 2°C with 50% ± 20% relative humidity using at least 10-15 air changes/h of 100% conditioned fresh air with a 12-h 0600 (light) to 1800 (dark) full-spectrum lighting cycle. Mouse tails were tattooed for individual identification during acclimation. B6D2F1/J female mice were randomly divided into 4 groups: 1) sham (N=10); 2) wound (N=10); 3) Radiation (N=20) ; 4) Radiation+wound (N=20).The sham-irradiated animals were treated in the same manner as the irradiated animals but not exposed to the radiation source. The experiments were performed twice with a half number of mice in each experiment. However, some mice in both RI and CI groups did not survive to day 30, resulting in fewer than 20 mice remaining in these groups by the study's end. The mice were euthanized 30 days after exposure to irradiation by exsanguination under isoflurane, and cervical dislocations were performed as a secondary physical method.

Radiation. Animals were subjected to irradiation as previously described [57]. Briefly, mice in the RI group received 9.5 Gy of ⁶⁰Co gamma photons, delivered at 0.4 Gy/min in the Uniformed Services University ⁶⁰Co facility. For the CI group, mice were exposed to 9.0 Gy of ⁶⁰Co, delivered at 0.4 Gy min^-1 because other insults are known to increase radiation sensitivity and the use of 9.5 Gy in the CI group resulted in excessive lethality (data not shown). Sham mice and wounded mice were handled the same way, but remained in the staging room outside the ⁶⁰Co facility. During irradiation, mice were kept awake in ventilated, acrylic plastic boxes with four compartments. The accuracy of the delivered doses was confirmed using an ionization chamber calibrated to measure the radiation dose to the midline soft tissue of the mice [57].

Wounding. Within 1 hr after irradiation, animals were anesthetized with isoflurane, and a wound was created 19±1.3 mm from the occipital bone and between the scapulae, using a punch [32]. Wounded and CI mice were injected i.p. with 0.5 ml saline containing 150 mg/kg acetaminophen (OFIRMEV injection, NDC 43825-102-01; Mallinckrodte Pharmaceuticals, Hazelwood, MO, USA) immediately after wounding to alleviate the pain. Sham mice and irradiated only mice received 0.5 ml saline i.p. The animal procedures are summarized in Figure 7.

Blood urea nitrogen (BUN) assay. Blood was collected via a cardiac puncture under isoflurane on day 30. BUN levels in serum were measured using the diacetyl monoxime method, as previously described [60,61]. Briefly, serum samples were diluted tenfold with deionized water. Proteins in the samples were precipitated by adding a 0.61 M trichloroacetic acid solution, and then separated by centrifuging for 10 minutes at room temperature, at 10,000 g. The resulting supernatants were mixed with a chromogen and incubated for 5 min at 80°C, and absorption measurements were taken with a microplate reader (Elx800, BioTek, Winooski, Vermont, United States) at 490 nm. The chromogen was created by freshly mixing Reagent A and Reagent B in a 2:1 ratio. Reagent A was prepared by dissolving 10 mg of ferric chloride in 10 ml of 1.48 M phosphoric acid, which was then mixed with 60 ml of distilled water and 30 ml of 5.52 M sulfuric acid. Reagent B consisted of 50 mg of diacetyl monoxime with 1 mg of thiosmicarbazide dissolved in 10 ml of distilled water [60,61].

Serum neutrophil gelatinase-associated lipocalin (NGAL) assay. Serum NGAL was detected using a kit from Abcam (ab199083) according to the manufacturer’s protocol.

Western blot analysis. The kidney cortex was homogenized in a lysis buffer consisting of 10 mM triethanolamine, 250 mM sucrose, pH 7.4 plus a protease inhibitor cocktail tablet (Roche, Catolog # 11784500), 2mM of NaF (MilliporeSigma, Catalog# S6776) and 2 mM of Na₃VO₄ (MilliporeSigma, Catalog# 450243) to inhibit phosphatases. The homogenates were centrifuged at 13000 rpm at 4⁰C for 8 min [62]. The protein concentrations in supernatants were measured with the BCA assay (Thermo Scientific, Product# 23228 and 1859078). After assay, samples were dissolved in an SDS loading buffer for immediate use or frozen at -80⁰C for future use. Equal quantities of protein samples (between 25-60 μg protein/lane) were loaded into 15 well or 17 well 4%–12% Bis-Tris gels (ThermoFisher, Catalog# NP0336BOX or NW04127BOX). After gel electrophoresis, the proteins were transferred onto nitrocellulose membranes (ThermoFisher, Catalog# LC2001). The membranes were soaked in blocking buffer (Odyssey, Part # 927-90001) at room temperature for 1 hour and the gels were stained with SimplyBlue^tm SafeStain (ThermoFisher, Catalog # LC6060) overnight to examine sample loadings. After soaking, the membranes were probed overnight with primary antibodies typically at 1:500 or 1:1000 dilution at 4°C. The membranes were then washed with PBS+0.1% Tween-20 and probed with an Alexa Fluor® secondary antibody for 1 hour at room temperature then examined with Odyssey, an infrared imaging scanner (Li-Cor). The data is presented without normalization, as stained gels were used to confirm equal sample loading. Reserving the first lane for protein markers, the maximum number of samples we could accommodate was 14 or 16. Protein samples were loaded into gels according to the randomly assigned numbers of mice, without any selection bias. If a significant difference in a protein abundance was observed in the first Western blot analysis, the experiment was repeated once with reproducible results presented. If there was no clear trend for a significant difference of protein levels in the first Western blot analysis, the experiment was stopped. The inconsistencies in sample numbers could arise from several factors. Occasionally, a bubble formed in the first well, or a distortion occurred in the last well, preventing their inclusion in the analysis. The data presented in the manuscript represents only a subset of the analyses we conducted. At times, we faced a shortage of samples for certain groups but proceeded with the Western blot analysis regardless. These results are included in the manuscript. The primary antibodies against proteins are listed as follows: Kidney injury molecule-1 (KIM-1, Invitrogen, PA520244), STAT1-Y701-P (Cell Signaling, 9167), STAT1 (Cell Signaling, 9176), STAT3-Y705-P (Cell Signaling, 9145S), STAT3 (Cell Signaling, 9139S), SHP-1-S591(ECM Biosciences, SP1531), SHP-1 (Santa Cruz, sc-287), p38-P (Cell Signaling, 9215S), p38 (Upstate, 05-454), MnSOD (Upstate, 06-984), Cu/ZnSOD (Upstate, 07-403), Peroxiredoxin 1 (PRDX) (Cell Signaling, 8732S), HO-1 (Cell Signaling, 43966S), p53 (Cell Signaling, 2524), AKT-S473 (Cell Signaling, 4060S), AKT (Cell Signaling, 2920S), GSK-3β-S9 (Cell Signaling, 9336), and GSK-3β (Cell Signaling, 9832).

qPCR. Quantitative PCR (qPCR) analysis of mRNA abundance were performed as described previously [60]. Briefly, total RNA was isolated with the TRIzol kit (ThermoFisher, Ref# 15596026) and measured with NanoDrop 8000 Spectrophotometer (ThermoFisher). cDNAs were synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Part# 4368814). mRNAs were measured using the Fast SYBR Green Master Mix (Applied Biosystems, Ref# 4385612) in a Cycler 480 (Roche) and normalized to a housekeeping gene, L32. Fold difference was calculated as previously described [60]. The primers used are listed in Table 1.

Statistical analysis. Data are expressed as means ± standard errors. All analyses were normalized to the average value of the respective control groups. Shapiro-Wilk normality test and Q-Q plot revealed normal distributions of our data. Statistical analyses were performed using a non-paired two-tailed t-test with Microsoft Excel. Two-way ANOVA could not be performed because two different doses of radiation were used in the RI and CI groups: 9.5 Gy and 9.0 Gy, respectively. A p value less than 0.05 was considered significant.

5. Conclusions

In conclusion, our findings have highlighted the complexity of the body's response to RI and CI, revealing both unique and shared pathways in inflammation, oxidative stress, and cellular survival mechanisms. These intriguing insights have laid the groundwork for understanding the mechanisms underlying delayed functional impairment in the kidney and open new targets such signaling molecules for future research into therapeutic strategies to protect renal function in the events of nuclear attacks and accidents, with a particular focus on the distinct and common molecular pathways activated by RI and CI.